Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is a multifunctional enzyme that serves as major target for the development of drugs to treat infection with this virus. Both DNA polymerase and ribonuclease H (RNase H) activities are essential for conversion of the single-stranded RNA genome into double-stranded DNA. However, all clinically used RT inhibitors are directed against the polymerase active site of the enzyme, while drugs that block specifically RNase H activity have yet to be developed. The primary goal of this proposal is to establish novel tools for the study of RNase H inhibition. We have previously developed site-specific footprinting techniques that allow us to precisely determine the position of HIV-1 RT on its primer/template substrate. We will further explore this technology in order to study changes in the trajectory of the bound nucleic acid substrate. We hypothesize that compounds that bind specifically to the RNase H domain can induce such changes that produce an unfavorable interaction between the enzyme and its RNA substrate. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]